Received for publication June 27, 2005.
Revised September 23, 2005.
Accepted for publication October 11, 2005.

FLAVOPIRIDOL AND HISTONE DEACETYLASE INHIBITORS PROMOTE MITOCHONDRIAL INJURY AND CELL DEATH IN HUMAN LEUKEMIA CELLS OVEREXPRESSING BCL-2

Abstract

Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and histone deacetylase (HDAC) inhibitors (SAHA and sodium butyrate; SB) were examined in human leukemia cells (U937, HL-60) ectopically expressing Bcl-2/Bcl-x_L and in primary AML cells. Co-administration of flavopiridol with HDAC inhibitors synergistically potentiated mitochondrial damage (cytochrome c, Smac/DIABLO, and AIF release), caspase activation, PARP degradation, and cell death in both wild type and Bcl-2- or Bcl-x_L-overexpressing cells and induced a pronounced loss of clonogenicity. In contrast, Bcl-2 and Bcl-x_L largely blocked these events in cells exposed to the cytotoxic agent ara-C. Enforced expression of dominant-negative FADD failed to protect cells from the flavopiridol/HDACI regimen, arguing against the involvement of the receptor pathway in lethality. Ectopic expression of a phosphorylation loop-deleted Bcl-2 or Bcl-2 lacking the serine₇₀ phosphorylation site, which dramatically protected cells from ara-C lethality, delayed but did not prevent flavopiridol/HDAC inhibitor-induced mitochondrial injury, cell death, or loss of clonogenicity. Ectopic expression of Bcl-2 or Bcl-x_L was also unable to prevent the flavopiridol/HDACI regimen from inducing a conformational change in and mitochondrial translocation of Bax, nor did it attenuate Bax dimerization. Collectively, these findings indicate that in contrast to certain conventional cytotoxic agents such as ara-C, overexpression of Bcl-2 or Bcl-x_L are largely ineffective in preventing perturbations in Bax, mitochondrial injury, and cell death in human leukemia cells subjected to simultaneous CDK and HDAC inhibition. They also raise the possibility that a strategy combining CDK and HDAC inhibitors may be effective against drug-resistant leukemia cells overexpressing Bcl-2 or Bcl-x_L.

Key words: Mechanisms of cell killing/apoptosis, Resistance

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